| As an essential trace element,iron is involved in physiological processes such as cell metabolism,DNA synthesis,and hemoglobin synthesis.Excessive or insufficient intake of iron can adversely affect the organism.The NRC（1994）standard for laying hens stipulated that the iron content in feed formulations for brown-shelled hens should reach 56-75 mg/kg,and this standard reaches 60-80 mg/kg in China.In the production,iron is generally added in the basic ration to improve the production performance of laying hens.However,some mineral additives in China,the addition of Fe SO4 between 300-3000 mg/kg,resulting in a feed containing much more iron than the actual requirement.When the iron supply exceeds requirement,it might causes iron overload in the body,which is toxic to the organisms,and the excess iron excreted in the feces may also leads to environmental pollution via excreta.The liver is the main regulator of iron homeostasis in the body,and disorders of iron metabolism lead to excess free iron participating in the Fenton reaction,generating large amounts of reactive oxygen species,inducing lipid peroxidation reactions that damage cell membrane structures and eventually lead to liver injury.In order to reveal the mechanism of liver injury in chickens caused by iron overload,we conducted the following studies from the perspectives of oxidative stress,glucose and lipid metabolism disorders,bile acid metabolism disorders,and inflammation.Sixty 1-day-old Hyland brown laying hens were randomly divided into Control group（C,fed with basal diet containing 139 mg/kg iron）,Low group（L,fed with basal diet supplemented with additional 450 mg/kg iron）,Medium group（M,fed with basal diet supplemented with additional 750 mg/kg iron）,and High group（H,fed with basal diet supplemented with additional 1050 mg/kg iron）.Blood and liver tissue were collected after 7 weeks of the experiment,and the results were as follows.Firstly,the serum iron and serum ferritin levels in the serum of laying hens were detected with commercial kits.The results showed that the serum iron content increased with the increase of dietary iron level,indicating a dose-dependent increase of the body iron content with dietary iron level.Iron content in the liver was examined by Prussian blue staining and tissue iron assay kit.The expression levels of hepatic iron metabolism-related proteins were detected by western blot,including TFRC,DMT1,FPN1,FTH,FTL,IREB2,and FBXL5.The results showed that high iron levels in the diet led to increased hepatic iron content,decreased iron absorption,and increased storage and excretion,indicating that high iron levels in the diet led to hepatic iron overload.Secondly,the histological observation of liver pathology combined with the detection of liver function-related indicators in serum suggested that iron overload led to an impaired liver tissue structure.Elevated liver injury indicator enzymes,impaired protein synthesis and bilirubin metabolism in serum indicated that iron overload led to liver injury.The liver oxidative stress markers,including T-SOD,CAT,and MDA,were detected by the commercial kits.The results showed that iron overload decreased antioxidant enzymes and increased lipid peroxidation products in the liver,indicating that iron overload induced oxidative stress in the liver.Thirdly,real-time quantitative PCR was used to detect the expression levels of hepatic glucose and lipid metabolism-related genes,including PPAR-α,LPL,CPT1A,PNPLA2,SCD-1,FASN,GPAM,PPAR-γ,GLUT2,GLUT8,G6PC,and PEPCK.The results showed that iron overload increased hepatic lipid synthesis and gluconeogenesis,while decreased lipolysis,indicating that iron overload led to disorders of hepatic glucolipid metabolism.The expression levels of genes related to hepatic bile acid metabolism,including CYP7A1,CYP7B1,CYP8B1,BAAT,FXR,BSEP,and NTCP were detected by real-time quantitative PCR.The results showed that iron overload decreased hepatic bile acid synthesis but increased output,indicating that iron overload caused disorders of hepatic bile acid metabolism.Fourthly,western blot was used to detect the expression levels of proteins involving inflammation,including i NOS,NF-κB,TNF-α,and COX-2.And real-time quantitative PCR was used to detect the expression levels of inflammatory cytokines,including IL-1β,IL-6,IL-10,and IL-18 in the liver.The results showed that iron overload elevated expression levels of pro-inflammation-related proteins and genes in the liver,indicating that iron overload triggered an inflammatory response in the liver.In conclusion,high levels of iron in the diet results in an iron overload of the liver,which induces oxidative stress,disturbance of hepatic glucose and lipid metabolism,disorder of bile acid metabolism,and inflammation. |
